DESCRIPTION: Dr. Scarpelli has developed a model of pancreatic cancer using carcinogen treatment of Syrian golden hamster (SGH) pancreatic cells in vitro. In this proposal, he plans to continue studies on the molecular and genetic changes induced by chemical carcinogens both in vivo and in vitro, and he will begin to develop an in vitro carcinogenesis model for human pancreatic duct cells. To accomplish these goals, he will isolate SGH genes homologous to the human genes altered in pancreatic cancer; he will use the new method of differential display of mRNA to search for unidentified genes that may play a pathogenetic role; he will determine if NNK, BP, or Glu-P-1 are capable of transforming SGH and rat pancreatic duct cells in vitro; and he will immortalize human pancreatic duct cells in vitro. In previous work on this grant, he has used SGH cells in vitro to explore the action of BOP, MNU, and HPNU in pancreatic carcinogenesis, in studies of both the mutagenic activity of these agents and alterations in their genetic targets. Two of the early original aims of the grant, to characterize two secreted proteins from SGH cells, and to determine whether BOP could mutagenize individual duct cells, were largely abandoned because of technical difficulties. In particular, Dr. Scarpelli determined that nude mice injected subcutaneously with duct cells co-cultured with hamster hepatocytes and treated with BOP in vitro developed tumors at various doses of BOP. When duct cells were not co-cultured, tumors did not develop. However, only the lowest dose of carcinogen was used and few experiments could be done, as he pointed out. He then attempted to determine whether the DNA of hamster pancreatic duct cells is more susceptible to damage by MNU than that of rat pancreatic duct cells. The time course of unscheduled DNA synthesis as a function of MNU concentration was found to be about 1.5-2 fold greater in HDC cells than in rat pancreatic duct cells (RDC). Similarly, tritiated thymidine incorporation into nuclear DNA was 5-10 fold higher in HDC than in RDC cells. Because of the difficulties of treating individual duct cells with BOP, he used MNU and HPNU in subsequent experiments. After treatment with various dosages of each agent, he selected initiated cells by growing them in media deficient for serum and EGF. Cells treated in this manner died, and surviving cells eventually formed patchy monolayers with numerous mitoses and a clear zone between cells. These treated and selected cells formed tumors in nude mice at low and high doses of MNU, while treated nonselected cells also formed tumors at high doses of MNU. By examining K-ras in these tumors, he found that long treatment with MNU led to codon 12 mutations, and short treatment led to codon 13 mutations. In contrast, HPNU showed differences depending upon the dose used (only short treatments were used). K-ras showed codon 12 mutations after low doses, and codon 13 mutations after high doses. Using PCR, exons 5 through 8 of the p53 gene were cloned from SGH cells, and a polyclonal antibody was obtained from a bacterial fusion protein. He showed a mutation in p53 in a transplantable BOP- induced tumor. SSCP was done on some MNU- and BOP-treated cells, showing a conformational change in exon 7, but this change was not seen in the nude mouse tumors derived from these cells. Finally, a small fragment of the hamster DCC gene was cloned by PCR, as well as a fragment of the hamster mdm-2 gene. There were 4 peer-reviewed publications during the grant period, in print or accepted for publication, including 2 in Cancer Research, 1 in Pancreas, and 1 in Carcinogenesis.